A Novel Environmental Azole Resistance Mutation in Aspergillus fumigatus and a Possible Role of Sexual Reproduction in Its Emergence

Abstract

This study investigated the dynamics of Aspergillus fumigatus azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR₄₆/Y121F/T289A genotype, 2% (1/45) harbored the TR₄₆/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR₄₆³/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR₄₆³ mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR₄₆ repeats. Importantly, in the laboratory, we recovered the TR₄₆³ mutation from a sexual cross between two TR₄₆ isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases.IMPORTANCE Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in Aspergillus fumigatus In this article, we show the dominance of azole-resistant A. fumigatus in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant A. fumigatus isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of Aspergillus diseases.

Keywords: Aspergillus fumigatus; ascospores; azole resistance; compost heap; conidiospores; hot spot for resistance development; novel mutation; sexual reproduction.

FIG 1

(A) Year in which for the first time since 1998 each of the different azole-resistant A. fumigatus isolates with TR promoter mutations was observed in The Netherlands. (B) Genotype illustration of the azole-resistance mutations in the cyp51A gene (promoter and coding region).

FIG 2

Locations and positions of samples taken from azole-free compost in Wageningen (W1 to W6 [30 cm apart from top to bottom]) and azole-containing compost in Hillegom (H1 to H6).

FIG 3

A. fumigatus CFU density (CFU per gram of compost) before and after heat shock in samples taken at different positions in an azole-free compost heap (W1 to W6 [dark blue bars]) and an azole-containing compost heap (H1 to H6 [light blue bars]).

FIG 4

Azole-resistance assay based on MGR of the TR₃₄, TR₄₆, and TR₄₆³ variants against voriconazole (Vor), itraconazole (Itr), and posaconazole (Pos). MEA was supplemented with 2 mg/liter of Vor and Itr, respectively, with Pos at 0.5 mg/liter. None, without any azoles. Error bars indicate the standard error of the mean (SEM). The MGR was determined by averaging the colony diameters (in millimeters) as measured in two randomly chosen perpendicular directions.

FIG 5

Gene expression levels of cyp51A in TR variants (WT TR₃₄, TR₄₆, and TR₄₆³). Bars show averages from nine replicates, and error bars indicate the standard error of the mean (SEM).

FIG 6

A possible scenario indicating how unequal crossing over in a sexual cross between two strains with a double repeat TR₄₆ can result in a rare meiotic recombinant with the triple repeat TR₄₆³. Yellow boxes represent the tandem repeats in the promoter region of cyp51A gene, and red ovals represent point mutations in the coding region of the cyp51A gene. The bent blue line indicates unequal crossover.